Abstract
Posttranslational modification of proteins by small ubiquitin-like modifier (SUMO) is required for survival of virtually all eukaryotic organisms. Attachment of SUMO to target proteins is catalyzed by SUMO E2 conjugase. All haploid or diploid eukaryotes studied to date possess a single indispensable SUMO conjugase. We report here the unanticipated isolation of a Chlamydomonas reinhardtii (mutant5 [mut5]). in which the previously identified SUMO conjugase gene C. reinhardtii ubiquitin-conjugating enzyme9 (CrUBC9) is deleted. This surprising mutant is viable and unexpectedly, displays a pattern of protein SUMOylation at 25°C that is essentially identical to wild-type cells. However, unlike wild-type cells, mut5 fails to SUMOylate a large set of proteins in response to multiple stress conditions, a failure that results in a markedly reduced tolerance or complete lack of tolerance to these stresses. Restoration of expected stress-induced protein SUMOylation patterns as well as normal stress tolerance phenotypes in mut5 cells complemented with a CrUBC9 gene shows that CrUBC9 is an authentic SUMO conjugase and, more importantly, that SUMOylation is essential for cell survival under stress conditions. The presence of bona fide SUMOylated proteins in the mut5 mutant at 25°C can only be explained by the presence of at least one additional SUMO conjugase in C. reinhardtii, a conjugase tentatively identified as CrUBC3. Together, these results suggest that, unlike all other nonpolyploid eukaryotes, there are at least two distinct and functional SUMO E2 conjugases in C. reinhardtii, with a clear division of labor between the two sets: One (CrUBC9) is involved in essential stress-induced SUMOylations, and one (CrUBC3) is involved in housekeeping SUMOylations.
Highlights
Plant Science Innovation, Center for SUMOylation by a Stress-Specific Small UbiquitinLike Modifier E2 Conjugase Is Essential for Survival of Chlamydomonas reinhardtii under Stress Conditions
Because in virtually every other haploid or diploid organism studied, a single small ubiquitin-like modifier (SUMO) E2 conjugase had been identified, it was presumed that C. reinhardtii ubiquitinconjugating enzyme9 (CrUBC9) was likely the only SUMO E2 conjugase present in the C. reinhardtii genome and that it was essential for cell viability
Initial characterization of the SUMOylation system in C. reinhardtii included the identification of a putative SUMO E2 conjugase (CrUBC9) that showed in vitro SUMOylation activity (Wang et al, 2008)
Summary
Siz (E3 ligase) mutants in Arabidopsis show increased tolerance to salt stress (Miura et al, 2011), whereas the SUMO protease (isopeptidase) double mutant, ots ots ( for overly sensitive to salt overly sensitive to salt2) shows extreme sensitivity to salt (Conti et al, 2008) Another indirect indication of the role of SUMOylation in the response of plants to stress is the recent observation linking changing levels of SUMOylation to changing levels of salicylic acid (Villajuana-Bonequi et al, 2014). Because in virtually every other haploid or diploid organism studied, a single SUMO E2 conjugase had been identified, it was presumed that CrUBC9 was likely the only SUMO E2 conjugase present in the C. reinhardtii genome and that it was essential for cell viability.
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